IcsA autotransporter passenger promotes increased fusion protein expression on the cell surface

Autotransporters are attractive cell surface display vehicles as they lack complex adaptor proteins necessary for protein export. Recent reports have suggested that the native effector domain (alpha domain) and translocation domain (beta domain) interact with each other to drive translocation of the effector domain to the outer membrane.

In this report we compared the expression, surface localisation and folding of TEM-1 beta-lactamase (Bla) and maltose binding protein (MalE or MBP) fused to either full length Shigella flexneri IcsA (IcsA) autotransporter or to the beta domain alone (IcsAbeta) to determine the contribution of the native IcsA alpha domain in presenting the fusion proteins on the surface of E. coli K-12 UT5600 ([increment]ompT).

Results: Expression of IcsA-Bla was greater than IcsAbeta-Bla.

High levels of IcsA-MalE were detected but IcsAbeta-MalE was not expressed. All fusion proteins other than IcsAbeta-MalE were localised to the outer membrane and were detected on the surface of UT5600 via immunofluorescence microscopy.

All bacteria expressing IcsA-MalE were labelled with both alpha-IcsA and alpha-MBP. UT5600 expressing IcsAbeta-MalE was not labelled with alpha-MBP.

A third of UT5600 expressing IcsA-Bla were detectable with alpha-Bla but only 5% of UT5600 (IcsAbeta-Bla) were labelled with alpha-Bla. The correct folding of the Bla moiety when fused to IcsA and IcsAbeta was also retained as UT5600 expressing either fusion protein exhibited a decreased zone of inhibition in the presence of ampicillin.

UT5600 expressing IcsA-Bla was more resistant compared to UT5600 expressing IcsAbeta-Bla.

Conclusions: The export mechanism of autotransporters is not well understood but accumulating evidence suggest a critical role for the native effector or alpha domain in facilitating its own export via interactions with the translocation or beta domain. This is the first report directly comparing expression of heterologous proteins fused to the full length IcsA autotransporter and fusion to the beta domain alone.

Protein expression and surface presentation of the fusion proteins were dramatically improved when fused to IcsA rather than IcsAbeta. Future studies involved in designing autotransporters as cell surface display vehicles would benefit from including the native alpha domain.

This work also provides further evidence for a key interaction between the autotransporter alpha and beta domains.

Author: Mabel LumRenato Morona
Credits/Source: Microbial Cell Factories 2012, 11:20

Published on: 2012-02-07

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